Caveolin-1 Modulates HIV-1 Envelope-Induced Bystander Apoptosis through gp41

Human immunodeficiency virus (HIV) envelope (Env)-mediated bystander apoptosis is known to cause the progressive, severe, and irreversible loss of CD4⁺ T cells in HIV-1-infected patients. Env-induced bystander apoptosis has been shown to be gp41 dependent and related to the membrane hemifusion between envelope-expressing cells and target cells. Caveolin-1 (Cav-1), the scaffold protein of specific membrane lipid rafts called caveolae, has been reported to interact with gp41. However, the underlying pathological or physiological meaning of this robust interaction remains unclear. In this report, we examine the interaction of cellular Cav-1 and HIV gp41 within the lipid rafts and show that Cav-1 modulates Env-induced bystander apoptosis through interactions with gp41 in SupT1 cells and CD4⁺ T lymphocytes isolated from human peripheral blood. Cav-1 significantly suppressed Env-induced membrane hemifusion and caspase-3 activation and augmented Hsp70 upregulation. Moreover, a peptide containing the Cav-1 scaffold domain sequence markedly inhibited bystander apoptosis and apoptotic signal pathways. Our studies shed new light on the potential role of Cav-1 in limiting HIV pathogenesis and the development of a novel therapeutic strategy in treating HIV-1-infected patients.HIV infection causes a progressive, severe, and irreversible depletion of CD4⁺ T cells, which is responsible for the development of AIDS (9). The mechanism through which HIV infection induces cell death involves a variety of processes (58). Among these processes, apoptosis is most likely responsible for T-cell destruction in HIV-infected patients (33), because active antiretroviral therapy has been associated with low levels of CD4⁺ T-cell apoptosis (7), and AIDS progression was shown previously to correlate with the extent of immune cell apoptosis (34). Importantly, bystander apoptosis of uninfected cells was demonstrated to be one of the major processes involved in the destruction of immune cells (58), with the majority of apoptotic CD4⁺ T cells in the peripheral blood and lymph nodes being uninfected in HIV patients (22).Binding to uninfected cells or the entry of viral proteins released by infected cells is responsible for the virus-mediated killing of innocent-bystander CD4⁺ T cells (2-4, 9, 65). The HIV envelope glycoprotein complex, consisting of gp120 and gp41 subunits expressed on an HIV-infected cell membrane (73), is believed to induce bystander CD4⁺ T-cell apoptosis (58). Although there is a soluble form of gp120 in the blood, there is no conclusive agreement as to whether the concentration is sufficient to trigger apoptosis (57, 58). The initial step in HIV infection is mediated by the Env glycoprotein gp120 binding with high affinity to CD4, the primary receptor on the target cell surface, which is followed by interactions with the chemokine receptor CCR5 or CXCR4 (61). This interaction triggers a conformational change in gp41 and the insertion of its N-terminal fusion peptide into the target membrane (30). Next, a prehairpin structure containing leucine zipper-like motifs is formed by the two conserved coiled-coil domains, called the N-terminal and C-terminal heptad repeats (28, 66, 70). This structure quickly collapses into a highly stable six-helix bundle structure with an N-terminal heptad repeat inside and a hydrophobic C-terminal heptad repeat outside (28, 66, 70). The formation of the six-helix bundle leads to a juxtaposition and fusion with the target cell membrane (28, 66, 70). The fusogenic potential of HIV Env is proven to correlate with the pathogenesis of both CXCR4- and CCR5-tropic viruses by not only delivering the viral genome to uninfected cells but also mediating Env-induced bystander apoptosis (71). Initial infection is dominated by the CCR5-tropic strains, with the CXCR4-tropic viruses emerging in the later stages of disease (20). Studies have shown that CXCR4-tropic HIV-1 triggers more depletion of CD4⁺ T cells than CCR5-tropic strains (36).Glycolipid- and cholesterol-enriched membrane microdomains, termed lipid rafts, are spatially organized plasma membranes and are known to have many diverse functions (26, 53). These functions include membrane trafficking, endocytosis, the regulation of cholesterol and calcium homeostasis, and signal transduction in cellular growth and apoptosis. Lipid rafts have also been implicated in HIV cell entry and budding processes (19, 46, 48, 51). One such organelle is the caveola, which is a small, flask-shaped (50 to 100 nm in diameter) invagination in the plasma membrane (5, 62). The caveola structure, which is composed of proteins known as caveolins, plays a role in various functions by serving as a mobile platform for many receptors and signal proteins (5, 62). Caveolin-1 (Cav-1) is a 22- to 24-kDa major coat protein responsible for caveola assembly (25, 47). This scaffolding protein forms a hairpin-like structure and exists as an oligomeric complex of 14 to 16 monomers (21). Cav-1 has been shown to be expressed by a variety of cell types, mostly endothelial cells, type I pneumocytes, fibroblasts, and adipocytes (5, 62). In addition, Cav-1 expression is evident in immune cells such as macrophages and dendritic cells (38, 39). However, Cav-1 is not expressed in isolated thymocytes (49). Furthermore, Cav-1 and caveolar structures are absent in human or murine T-cell lines (27, 41, 68). Contrary to this, there has been one report showing evidence of Cav-1 expression in bovine primary cell subpopulations of CD4⁺, CD8⁺, CD21⁺, and IgM⁺ cells with Cav-1 localized predominantly in the perinuclear region (38). That report also demonstrated a membrane region staining with Cav-1-specific antibody of human CD21⁺ and CD26⁺ peripheral blood lymphocytes (PBLs). Recently, the expression of Cav-1 in activated murine B cells, with a potential role in the development of a thymus-independent immune response, was also reported (56). It remains to be determined whether Cav-1 expression is dependent on the activation state of lymphocytes. For macrophages, however, which are one of the main cell targets for HIV infection, Cav-1 expression has been clearly documented (38).The scaffolding domain of Cav-1, located in the juxtamembranous region of the N terminus, is responsible for its oligomerization and binding to various proteins (5, 62, 64). It recognizes a consensus binding motif, ΦXΦXXXXΦ, ΦXXXXΦXXΦ, or ΦXΦXXXXΦXXΦ, where Φ indicates an aromatic residue (F, W, or Y) and X indicates any residue (5, 62, 64). A Cav-1 binding motif (WNNMTWMQW) has been identified in the HIV-1 envelope protein gp41 (42, 43). Cav-1 has been shown to associate with gp41 by many different groups under various circumstances, including the immunoprecipitation of gp41 and Cav-1 in HIV-infected cells (42, 43, 52). However, the underlying pathological or physiological functions of this robust interaction between Cav-1 and gp41 remain unclear.Here, we report that the interaction between Cav-1 and gp41 leads to a modification of gp41 function, which subsequently regulates Env-induced T-cell bystander apoptosis. Moreover, we show that a peptide containing the Cav-1 scaffold domain sequence is capable of modulating Env-induced bystander apoptosis, which suggests a novel therapeutic application for HIV-1-infected patients.     

Selective Expression of Human Immunodeficiency Virus Nef in Specific Immune Cell Populations of Transgenic Mice Is Associated with Distinct AIDS-Like Phenotypes

We previously reported that CD4C/human immunodeficiency virus (HIV)^Nef transgenic (Tg) mice, expressing Nef in CD4⁺ T cells and cells of the macrophage/dendritic cell (DC) lineage, develop a severe AIDS-like disease, characterized by depletion of CD4⁺ T cells, as well as lung, heart, and kidney diseases. In order to determine the contribution of distinct populations of hematopoietic cells to the development of this AIDS-like disease, five additional Tg strains expressing Nef through restricted cell-specific regulatory elements were generated. These Tg strains express Nef in CD4⁺ T cells, DCs, and macrophages (CD4E/HIV^Nef); in CD4⁺ T cells and DCs (mCD4/HIV^Nef and CD4F/HIV^Nef); in macrophages and DCs (CD68/HIV^Nef); or mainly in DCs (CD11c/HIV^Nef). None of these Tg strains developed significant lung and kidney diseases, suggesting the existence of as-yet-unidentified Nef-expressing cell subset(s) that are responsible for inducing organ disease in CD4C/HIV^Nef Tg mice. Mice from all five strains developed persistent oral carriage of Candida albicans, suggesting an impaired immune function. Only strains expressing Nef in CD4⁺ T cells showed CD4⁺ T-cell depletion, activation, and apoptosis. These results demonstrate that expression of Nef in CD4⁺ T cells is the primary determinant of their depletion. Therefore, the pattern of Nef expression in specific cell population(s) largely determines the nature of the resulting pathological changes.The major cell targets and reservoirs for human immunodeficiency virus type 1 (HIV-1)/simian immunodeficiency virus (SIV) infection in vivo are CD4⁺ T lymphocytes and antigen-presenting cells (macrophages and dendritic cells [DC]) (21, 24, 51). The cell specificity of these viruses is largely dependent on the expression of CD4 and of its coreceptors, CCR5 and CXCR-4, at the cell surface (29, 66). Infection of these immune cells leads to the severe disease, AIDS, showing widespread manifestations, including progressive immunodeficiency, immune activation, CD4⁺ T-cell depletion, wasting, dementia, nephropathy, heart and lung diseases, and susceptibility to opportunistic pathogens, such as Candida albicans (1, 27, 31, 37, 41, 82, 93, 109). It is reasonable to assume that the various pathological changes in AIDS result from the expression of one or many HIV-1/SIV proteins in these immune target cells. However, assigning the contribution of each infected cell subset to each phenotype has been remarkably difficult, despite evidence that AIDS T-cell phenotypes can present very differently depending on the strains of infecting HIV-1 or SIV or on the cells targeted by the virus (4, 39, 49, 52, 72). For example, the T-cell-tropic X4 HIV strains have long been associated with late events and severe CD4⁺ T-cell depletion (22, 85, 96). However, there are a number of target cell subsets expressing CD4 and CXCR-4, and identifying which one is responsible for this enhanced virulence has not been achieved in vivo. Similarly, the replication of SIV in specific regions of the thymus (cortical versus medullary areas), has been associated with very different outcomes but, unfortunately, the critical target cells of the viruses were not identified either in these studies (60, 80). The task is even more complex, because HIV-1 or SIV can infect several cell subsets within a single cell population. In the thymus, double (CD4⁻ CD8⁻)-negative (DN) or triple (CD3⁻ CD4⁻ CD8⁻)-negative (TN) T cells, as well as double-positive (CD4⁺ CD8⁺) (DP) T cells, are infectible by HIV-1 in vitro (9, 28, 74, 84, 98, 99, 110) and in SCID-hu mice (2, 5, 91, 94). In peripheral organs, gut memory CCR5⁺ CD4⁺ T cells are primarily infected with R5 SIV, SHIV, or HIV, while circulating CD4⁺ T cells can be infected by X4 viruses (13, 42, 49, 69, 70, 100, 101, 104). Moreover, some detrimental effects on CD4⁺ T cells have been postulated to originate from HIV-1/SIV gene expression in bystander cells, such as macrophages or DC, suggesting that other infected target cells may contribute to the loss of CD4⁺ T cells (6, 7, 32, 36, 64, 90).Similarly, the infected cell population(s) required and sufficient to induce the organ diseases associated with HIV-1/SIV expression (brain, heart, and kidney) have not yet all been identified. For lung or kidney disease, HIV-specific cytotoxic CD8⁺ T cells (1, 75) or infected podocytes (50, 95), respectively, have been implicated. Activated macrophages have been postulated to play an important role in heart disease (108) and in AIDS dementia (35), although other target cells could be infected by macrophage-tropic viruses and may contribute significantly to the decrease of central nervous system functions (11, 86, 97), as previously pointed out (25).Therefore, because of the widespread nature of HIV-1 infection and the difficulty in extrapolating tropism of HIV-1/SIV in vitro to their cell targeting in vivo (8, 10, 71), alternative approaches are needed to establish the contribution of individual infected cell populations to the multiorgan phenotypes observed in AIDS. To this end, we developed a transgenic (Tg) mouse model of AIDS using a nonreplicating HIV-1 genome expressed through the regulatory sequences of the human CD4 gene (CD4C), in the same murine cells as those targeted by HIV-1 in humans, namely, in immature and mature CD4⁺ T cells, as well as in cells of the macrophage/DC lineages (47, 48, 77; unpublished data). These CD4C/HIV Tg mice develop a multitude of pathologies closely mimicking those of AIDS patients. These include a gradual destruction of the immune system, characterized among other things by thymic and lymphoid organ atrophy, depletion of mature and immature CD4⁺ T lymphocytes, activation of CD4⁺ and CD8⁺ T cells, susceptibility to mucosal candidiasis, HIV-associated nephropathy, and pulmonary and cardiac complications (26, 43, 44, 57, 76, 77, 79, 106). We demonstrated that Nef is the major determinant of the HIV-1 pathogenicity in CD4C/HIV Tg mice (44). The similarities of the AIDS-like phenotypes of these Tg mice to those in human AIDS strongly suggest that such a Tg mouse approach can be used to investigate the contribution of distinct HIV-1-expressing cell populations to their development.In the present study, we constructed and characterized five additional mouse Tg strains expressing Nef, through distinct regulatory elements, in cell populations more restricted than in CD4C/HIV Tg mice. The aim of this effort was to assess whether, and to what extent, the targeting of Nef in distinct immune cell populations affects disease development and progression.     

Enhancement of human immunodeficiency virus (HIV)-specific CD8+ T cells in cerebrospinal fluid compared to those in blood among antiretroviral therapy-naive HIV-positive subjects

During untreated human immunodeficiency virus type 1 (HIV-1) infection, virus-specific CD8⁺ T cells partially control HIV replication in peripheral lymphoid tissues, but host mechanisms of HIV control in the central nervous system (CNS) are incompletely understood. We characterized HIV-specific CD8⁺ T cells in cerebrospinal fluid (CSF) and peripheral blood among seven HIV-positive antiretroviral therapy-naïve subjects. All had grossly normal brain magnetic resonance imaging and spectroscopy and normal neuropsychometric testing. Frequencies of epitope-specific CD8⁺ T cells by direct tetramer staining were on average 2.4-fold higher in CSF than in blood (P = 0.0004), while HIV RNA concentrations were lower. Cells from CSF were readily expanded ex vivo and responded to a broader range of HIV-specific human leukocyte antigen class I restricted optimal peptides than did expanded cells from blood. HIV-specific CD8⁺ T cells, in contrast to total CD8⁺ T cells, in CSF and blood were at comparable maturation states, as assessed by CD45RO and CCR7 staining. The strong relationship between higher T-cell frequencies and lower levels of viral antigen in CSF could be the result of increased migration to and/or preferential expansion of HIV-specific T cells within the CNS. This suggests an important role for HIV-specific CD8⁺ T cells in control of intrathecal viral replication.Human immunodeficiency virus type 1 (HIV-1) invades the central nervous system (CNS) early during primary infection (21, 30, 35), and proviral DNA persists in the brain throughout the course of HIV-1 disease (7, 25, 29, 47, 77, 83). Limited data from human and nonhuman primate studies suggest that little or no viral replication occurs in the brain during chronic, asymptomatic infection, based on the absence of demonstrable viral RNA or proteins (8, 85). In contrast, cognitive impairment affects approximately 40% of patients who progress to advanced AIDS without highly active antiretroviral therapy (21, 30, 35, 65). During HIV-associated dementia, there is active HIV-1 replication in the brain (23, 52, 61, 81), and viral sequence differences between cerebrospinal fluid (CSF) and peripheral tissues suggest distinct anatomic compartments of replication (18, 19, 22, 53, 75, 76, 78). Host mechanisms that control viral replication in the CNS during chronic, asymptomatic HIV-1 infection are incompletely understood.Anti-HIV CD8⁺ T cells are present in blood and peripheral tissues throughout the course of chronic HIV-1 infection (2, 14). Multiple lines of evidence support a critical role for these cells in controlling HIV-1 replication. During acute HIV-1 infection, the appearance of CD8⁺ T-cell responses correlates temporally with a decline in viremia (11, 43), and a greater proliferative capacity of peripheral blood HIV-specific CD8⁺ T cells correlates with better control of viremia (36, 54). In addition, the presence of certain major histocompatibility complex class I human leukocyte antigen (HLA) alleles, notably HLA-B*57, predicts slower progression to AIDS and death during chronic, untreated HIV-1 infection (55, 62). Finally, in the simian immunodeficiency virus (SIV) model, macaques depleted of CD8⁺ T cells experience increased viremia and rapid disease progression (39, 51, 67).Little is known regarding the role of intrathecal anti-HIV CD8⁺ T cells in HIV neuropathogenesis. Nonhuman primate studies have identified SIV-specific CD8⁺ T cells in the CNS early after infection (16, 80). Increased infiltration of SIV antigen-specific CD8⁺ T cells and cytotoxic T lymphocytes has been detected only in CSF of slow progressors without neurological symptoms (72). In chronically infected macaques with little or no SIV replication in the brain, the frequency of HIV-specific T cells was higher in CSF than in peripheral blood but did not correlate with the level of plasma viremia or CD4⁺ T-cell counts (56). Although intrathecal anti-HIV CD8⁺ T cells may help control viral replication, a detrimental role in the neuropathogenesis of HIV-1 has also been postulated (38). Immune responses contribute to neuropathogenesis in models of other infectious diseases, and during other viral infections cytotoxic T lymphocytes can worsen disease through direct cytotoxicity or release of inflammatory cytokines such as gamma interferon (IFN-γ) (3, 17, 31, 37, 42, 44, 71).We tested the hypothesis that quantitative and/or qualitative differences in HIV-specific CD8⁺ T-cell responses are present in CSF compared to blood during chronic, untreated HIV-1 infection. We characterized HIV-specific CD8⁺ T-cell responses in CSF among seven antiretroviral therapy-naïve adults with chronic HIV-1 infection, relatively high peripheral blood CD4⁺ T-cell counts, and low plasma HIV-1 RNA concentrations. We show that among these HIV-positive individuals with no neurological symptoms and with little or no HIV-1 RNA in CSF, frequencies of HIV-specific T cells are significantly higher in CSF than in blood. These CSF cells are at a state of differentiation similar to that of T cells in blood and are functionally competent for expansion and IFN-γ production. The higher frequency of functional HIV-specific CD8⁺ T cells in CSF, in the context of low or undetectable virus in CSF, suggests that these cells play a role in the control of intrathecal viral replication.     

Analysis of Human Immunodeficiency Virus Type 1 Viremia and Provirus in Resting CD4+ T Cells Reveals a Novel Source of Residual Viremia in Patients on Antiretroviral Therapy

Highly active antiretroviral therapy (HAART) can reduce human immunodeficiency virus type 1 (HIV-1) viremia to clinically undetectable levels. Despite this dramatic reduction, some virus is present in the blood. In addition, a long-lived latent reservoir for HIV-1 exists in resting memory CD4⁺ T cells. This reservoir is believed to be a source of the residual viremia and is the focus of eradication efforts. Here, we use two measures of population structure—analysis of molecular variance and the Slatkin-Maddison test—to demonstrate that the residual viremia is genetically distinct from proviruses in resting CD4⁺ T cells but that proviruses in resting and activated CD4⁺ T cells belong to a single population. Residual viremia is genetically distinct from proviruses in activated CD4⁺ T cells, monocytes, and unfractionated peripheral blood mononuclear cells. The finding that some of the residual viremia in patients on HAART stems from an unidentified cellular source other than CD4⁺ T cells has implications for eradication efforts.Successful treatment of human immunodeficiency virus type 1 (HIV-1) infection with highly active antiretroviral therapy (HAART) reduces free virus in the blood to levels undetectable by the most sensitive clinical assays (18, 36). However, HIV-1 persists as a latent provirus in resting, memory CD4⁺ T lymphocytes (6, 9, 12, 16, 48) and perhaps in other cell types (45, 52). The latent reservoir in resting CD4⁺ T cells represents a barrier to eradication because of its long half-life (15, 37, 40-42) and because specifically targeting and purging this reservoir is inherently difficult (8, 25, 27).In addition to the latent reservoir in resting CD4⁺ T cells, patients on HAART also have a low amount of free virus in the plasma, typically at levels below the limit of detection of current clinical assays (13, 19, 35, 37). Because free virus has a short half-life (20, 47), residual viremia is indicative of active virus production. The continued presence of free virus in the plasma of patients on HAART indicates either ongoing replication (10, 13, 17, 19), release of virus after reactivation of latently infected CD4⁺ T cells (22, 24, 31, 50), release from other cellular reservoirs (7, 45, 52), or some combination of these mechanisms. Finding the cellular source of residual viremia is important because it will identify the cells that are still capable of producing virus in patients on HAART, cells that must be targeted in any eradication effort.Detailed analysis of this residual viremia has been hindered by technical challenges involved in working with very low concentrations of virus (13, 19, 35). Recently, new insights into the nature of residual viremia have been obtained through intensive patient sampling and enhanced ultrasensitive sequencing methods (1). In a subset of patients, most of the residual viremia consisted of a small number of viral clones (1, 46) produced by a cell type severely underrepresented in the peripheral circulation (1). These unique viral clones, termed predominant plasma clones (PPCs), persist unchanged for extended periods of time (1). The persistence of PPCs indicates that in some patients there may be another major cellular source of residual viremia (1). However, PPCs were observed in a small group of patients who started HAART with very low CD4 counts, and it has been unclear whether the PPC phenomenon extends beyond this group of patients. More importantly, it has been unclear whether the residual viremia generally consists of distinct virus populations produced by different cell types.Since the HIV-1 infection in most patients is initially established by a single viral clone (23, 51), with subsequent diversification (29), the presence of genetically distinct populations of virus in a single individual can reflect entry of viruses into compartments where replication occurs with limited subsequent intercompartmental mixing (32). Sophisticated genetic tests can detect such population structure in a sample of viral sequences (4, 39, 49). Using two complementary tests of population structure (14, 43), we analyzed viral sequences from multiple sources within individual patients in order to determine whether a source other than circulating resting CD4⁺ T cells contributes to residual viremia and viral persistence. Our results have important clinical implications for understanding HIV-1 persistence and treatment failure and for improving eradication strategies, which are currently focusing only on the latent CD4⁺ T-cell reservoir.     

gp340 Promotes Transcytosis of Human Immunodeficiency Virus Type 1 in Genital Tract-Derived Cell Lines and Primary Endocervical Tissue

The human scavenger receptor gp340 has been identified as a binding protein for the human immunodeficiency virus type 1 (HIV-1) envelope that is expressed on the cell surface of female genital tract epithelial cells. This interaction allows such epithelial cells to efficiently transmit infective virus to susceptible targets and maintain viral infectivity for several days. Within the context of vaginal transmission, HIV must first traverse a normally protective mucosa containing a cell barrier to reach the underlying T cells and dendritic cells, which propagate and spread the infection. The mechanism by which HIV-1 can bypass an otherwise healthy cellular barrier remains an important area of study. Here, we demonstrate that genital tract-derived cell lines and primary human endocervical tissue can support direct transcytosis of cell-free virus from the apical to basolateral surfaces. Further, this transport of virus can be blocked through the addition of antibodies or peptides that directly block the interaction of gp340 with the HIV-1 envelope, if added prior to viral pulsing on the apical side of the cell or tissue barrier. Our data support a role for the previously described heparan sulfate moieties in mediating this transcytosis but add gp340 as an important facilitator of HIV-1 transcytosis across genital tract tissue. This study demonstrates that HIV-1 actively traverses the protective barriers of the human genital tract and presents a second mechanism whereby gp340 can promote heterosexual transmission.Through correlative studies with macaques challenged with simian immunodeficiency virus (SIV), the initial targets of infection in nontraumatic vaginal exposure to human immunodeficiency virus type 1 (HIV-1) have been identified as subepithelial T cells and dendritic cells (DCs) (18, 23, 31, 36-38). While human transmission may differ from macaque transmission, the existing models of human transmission remain controversial. For the virus to successfully reach its CD4⁺ targets, HIV must first traverse the columnar mucosal epithelial cell barrier of the endocervix or uterus or the stratified squamous barrier of the vagina or ectocervix, whose normal functions include protection of underlying tissue from pathogens. This portion of the human innate immune defense system represents a significant impediment to transmission. Studies have placed the natural transmission rate of HIV per sexual act between 0.005 and 0.3% (17, 45). Breaks in the epithelial barrier caused by secondary infection with other sexual transmitted diseases or the normal physical trauma often associated with vaginal intercourse represent one potential means for viral exposure to submucosal cells and have been shown to significantly increase transmission (reviewed in reference 11). However, studies of nontraumatic exposure to SIV in macaques demonstrate that these disruptions are not necessary for successful transmission to healthy females. This disparity indicates that multiple mechanisms by which HIV-1 can pass through mucosal epithelium might exist in vivo. Identifying these mechanisms represents an important obstacle to understanding and ultimately preventing HIV transmission.Several host cellular receptors, including DC-specific intercellular adhesion molecule-grabbing integrin, galactosyl ceramide, mannose receptor, langerin, heparan sulfate proteoglycans (HSPGs), and chondroitin sulfate proteoglycans, have been identified that facilitate disease progression through binding of HIV virions without being required for fusion and infection (2, 3, 12, 14, 16, 25, 29, 30, 43, 46, 50). These host accessory proteins act predominately through glycosylation-based interactions between HIV envelope (Env) and the host cellular receptors. These different host accessory factors can lead to increased infectivity in cis and trans or can serve to concentrate and expose virus at sites relevant to furthering its spread within the body. The direct transcytosis of cell-free virus through primary genital epithelial cells and the human endometrial carcinoma cell line HEC1A has been described (7, 9); this is, in part, mediated by HSPGs (7). Within the HSPG family, the syndecans have been previously shown to facilitate trans infection of HIV in vitro through binding of a specific region of Env that is moderately conserved (7, 8). This report also demonstrates that while HSPGs mediate a portion of the viral transcytosis that occurs in these two cell types, a significant portion of the observed transport occurs through an HSPG-independent mechanism. Other host cell factors likely provide alternatives to HSPGs for HIV-1 to use in subverting the mucosal epithelial barrier.gp340 is a member of the scavenger receptor cysteine-rich (SRCR) family of innate immune receptors. Its numerous splice variants can be found as a secreted component of human saliva (34, 41, 42) and as a membrane-associated receptor in a large number of epithelial cell lineages (22, 32, 40). Its normal cellular function includes immune surveillance of bacteria (4-6, 44), interaction with influenza A virus (19, 20, 32, 51) and surfactant proteins in the lung (20, 22, 33), and facilitating epithelial cell regeneration at sites of cellular inflammation and damage (27, 32). The secreted form of gp340, salivary agglutinin (SAG), was identified as a component of saliva that inhibits HIV-1 transmission in the oral pharynx through a specific interaction with the viral envelope protein that serves to agglutinate the virus and target it for degradation (34, 35, 41). Interestingly, SAG was demonstrated to form a direct protein-protein interaction with HIV Env (53, 54). Later, a cell surface-associated variant of SAG called gp340 was characterized as a binding partner for HIV-1 in the female genital tract that could facilitate virus transmission to susceptible targets of infection (47) and as a macrophage-expressed enhancer of infection (10).     

Induction of Robust Cellular and Humoral Virus-Specific Adaptive Immune Responses in Human Immunodeficiency Virus-Infected Humanized BLT Mice

The generation of humanized BLT mice by the cotransplantation of human fetal thymus and liver tissues and CD34⁺ fetal liver cells into nonobese diabetic/severe combined immunodeficiency mice allows for the long-term reconstitution of a functional human immune system, with human T cells, B cells, dendritic cells, and monocytes/macrophages repopulating mouse tissues. Here, we show that humanized BLT mice sustained high-level disseminated human immunodeficiency virus (HIV) infection, resulting in CD4⁺ T-cell depletion and generalized immune activation. Following infection, HIV-specific humoral responses were present in all mice by 3 months, and HIV-specific CD4⁺ and CD8⁺ T-cell responses were detected in the majority of mice tested after 9 weeks of infection. Despite robust HIV-specific responses, however, viral loads remained elevated in infected BLT mice, raising the possibility that these responses are dysfunctional. The increased T-cell expression of the negative costimulator PD-1 recently has been postulated to contribute to T-cell dysfunction in chronic HIV infection. As seen in human infection, both CD4⁺ and CD8⁺ T cells demonstrated increased PD-1 expression in HIV-infected BLT mice, and PD-1 levels in these cells correlated positively with viral load and inversely with CD4⁺ cell levels. The ability of humanized BLT mice to generate both cellular and humoral immune responses to HIV will allow the further investigation of human HIV-specific immune responses in vivo and suggests that these mice are able to provide a platform to assess candidate HIV vaccines and other immunotherapeutic strategies.An ideal animal model of human immunodeficiency virus (HIV) infection remains elusive. Nonhuman primates that are susceptible to HIV infection typically do not develop immunodeficiency (63), and although the simian immunodeficiency virus (SIV) infection of rhesus macaques has provided many critically important insights into retroviral pathogenesis (30), biological and financial considerations have created some limitations to the wide dissemination of this model. The great need for an improved animal model of HIV itself recently has been underscored by the disappointing results of human trials of MRKAd5, an adenovirus-based HIV type 1 (HIV-1) vaccine. This vaccine was not effective and actually may have increased some subjects'' risk of acquiring HIV (53). In the wake of these disappointing results, there has been increased interest in humanized mouse models of HIV infection (54). The ability of humanized mouse models to test candidate vaccines or other immunomodulatory strategies will depend critically on the ability of these mice to generate robust anti-HIV human immune responses.Mice have provided important model systems for the study of many human diseases, but they are unable to support productive HIV infection, even when made to express human coreceptors for the virus (7, 37, 52). A more successful strategy to humanize mice has been to engraft human immune cells and/or tissues into immunodeficient severe combined immunodeficiency (SCID) or nonobese diabetic (NOD)/SCID mice that are unable to reject xenogeneic grafts (39, 42, 57). Early versions of humanized mice supported productive HIV infection and allowed investigators to begin to address important questions in HIV biology in vivo (23, 40, 43-45). More recently, human cord blood or fetal liver CD34⁺ cells have been used to reconstitute Rag2^−/− interleukin-2 receptor γ chain-deficient (γ_c^−/−) and NOD/SCID/γ_c^−/− mice, resulting in higher levels of sustained human immune cell engraftment (27, 29, 61). These mice have allowed for stable, disseminated HIV infection (2, 4, 24, 65, 67), including mucosal transmission via vaginal and rectal routes (3). These mice recently have been used to demonstrate an important role for Treg cells in acute HIV infection (29) and to demonstrate that the T-cell-specific delivery of antiviral small interfering RNA is able to suppress HIV replication in vivo (31). These mice also have demonstrated some evidence of adaptive human immune responses, including the generation of HIV-specific antibody responses in some infected mice (2, 65), and some evidence of humoral and cell-mediated responses to non-HIV antigens or pathogens (24, 61). Most impressively, Rag2^−/− γ_c^−/− mice reconstituted with human fetal liver-derived CD34⁺ cells have generated humoral responses to dengue virus infection that demonstrated both class switching and neutralizing capacity (32). In spite of these advances, however, these models have not yet been reported to generate de novo HIV-specific cell-mediated immune responses, which are considered to be a crucial arm of host defense against HIV infection in humans.In contrast to humanized mouse models in which only human hematopoietic cells are transferred into immunodeficient mice, the surgical implantation of human fetal thymic and liver tissue has been performed in addition to the transfer of human hematopoietic stem cells (HSC) to generate mice in which human T cells are educated by autologous human thymic tissue rather than by the xenogeneic mouse thymus. Melkus and colleagues refer to mice they have reconstituted in this way as NOD/SCID-hu BLT (for bone marrow, liver, and thymus), or simply BLT, mice (41). We previously referred to mice that we have humanized in a similar way as NOD/SCID mice cotransplanted with human fetal thymic and liver tissues (Thy/Liv) and CD34⁺ fetal liver cells (FLC) (33, 60) but now adopt the designation BLT mice as well. BLT mice demonstrate the robust repopulation of mouse lymphoid tissues with functional human T lymphocytes (33, 41, 60) and can support the rectal and vaginal transmission of HIV (13, 59). Further, BLT mice demonstrate antigen-specific human immune responses against non-HIV antigens and/or pathogens (41, 60). The ability of these mice to generate human immune responses against HIV, however, has not yet been reported. In this study, we investigated whether the provision of autologous human thymic tissue in BLT mice generated by the cotransplantion of human fetal Thy/Liv tissues and CD34⁺ FLC would allow for the maturation of human T cells in humanized mice capable of providing improved cellular responses to HIV as well as providing adequate help for improved humoral responses. To describe the cells contributing to human immune responses in BLT mice, we also characterized the phenotypes of multiple subsets of T cells, B cells, dendritic cells (DCs), and monocytes/macrophages present in uninfected humanized mice. The generation of robust HIV-directed human cellular and humoral immune responses in these mice would further demonstrate the ability of humanized mice to provide a much needed platform for the evaluation of HIV vaccines and other novel immunomodulatory strategies.     

Reduction of Immune Activation with Chloroquine Therapy during Chronic HIV Infection

Increased levels of activated T cells are a hallmark of the chronic stage of human immunodeficiency virus (HIV) infection and are highly correlated with HIV disease progression. We evaluated chloroquine (CQ) as a potential therapy to reduce immune activation during HIV infection. We found that the frequency of CD38⁺ HLA-DR⁺ CD8 T cells, as well as Ki-67 expression in CD8 and CD4 T cells, was significantly reduced during CQ treatment. Our data indicate that treatment with CQ reduces systemic T-cell immune activation and, thus, that its use may be beneficial for certain groups of HIV-infected individuals.Chronic HIV infection is characterized by multifaceted systemic immune activation, including increased frequencies of activated T cells (9, 17) and increased turnover of T cells (5, 12, 18) that correlate directly with disease progression (8, 9). T-cell immune activation is also associated with lower gains in CD4 T-cell count in HIV-infected individuals even while they are on antiretroviral therapy (ART) that appears to suppress viral replication (10). Thus, therapies that reduce immune activation may be of benefit, particularly for such individuals. Three clinical studies have been conducted using hydroxychloroquine monotherapy for patients with HIV infection (6, 21, 22), and the studies showed that hydroxychloroquine-treated patients had decreased viral loads as well as decreased serum interleukin-6 (IL-6) levels, heightened levels of which are correlated with disease progression (13). However, these studies did not examine other parameters of immune activation. Chloroquine (CQ) is known to suppress immune activation by a number of mechanisms, including inhibition of intracellular toll-like receptor (TLR) signaling and inflammatory cytokine secretion (11, 19). In vitro, CQ has been shown to reduce HIV infection-induced T-cell immune activation (14). Here, we report results using samples from a clinical study of HIV-infected individuals treated with CQ monotherapy, where we examine multiple parameters of immune activation during the course of CQ treatment.     

Env-Expressing Autologous T Lymphocytes Induce Neutralizing Antibody and Afford Marked Protection against Feline Immunodeficiency Virus

The envelope (Env) glycoproteins of HIV and other lentiviruses possess neutralization and other protective epitopes, yet all attempts to induce protective immunity using Env as the only immunogen have either failed or afforded minimal levels of protection. In a novel prime-boost approach, specific-pathogen-free cats were primed with a plasmid expressing Env of feline immunodeficiency virus (FIV) and feline granulocyte-macrophage colony-stimulating factor and then boosted with their own T lymphocytes transduced ex vivo to produce the same Env and interleukin 15 (3 × 10⁶ to 10 × 10⁶ viable cells/cat). After the boost, the vaccinees developed elevated immune responses, including virus-neutralizing antibodies (NA). Challenge with an ex vivo preparation of FIV readily infected all eight control cats (four mock vaccinated and four naïve) and produced a marked decline in the proportion of peripheral CD4 T cells. In contrast, five of seven vaccinees showed little or no traces of infection, and the remaining two had reduced viral loads and underwent no changes in proportions of CD4 T cells. Interestingly, the viral loads of the vaccinees were inversely correlated to the titers of NA. The findings support the concept that Env is a valuable immunogen but needs to be administered in a way that permits the expression of its full protective potential.Despite years of intense research, a truly protective AIDS vaccine is far away. Suboptimal immunogenicity, inadequate antigen presentation, and inappropriate immune system activation are believed to have contributed to these disappointing results. However, several lines of evidence suggest that the control or prevention of infection is possible. For example, despite repeated exposures, some individuals escape infection or delay disease progression after being infected (1, 14, 15). Furthermore, passively infused neutralizing antibodies (NA) (28, 42, 51) or endogenously expressed NA derivatives (29) have been shown to provide protection against intravenous simian immunodeficiency virus challenge. On the other hand, data from several vaccine experiments suggest that cellular immunity is an important factor for protection (6, 32). Therefore, while immune protection against human immunodeficiency virus (HIV) and other lentiviruses appears feasible, the strategies for eliciting it remain elusive.Because of its crucial role in viral replication and infectivity, the HIV envelope (Env) is an attractive immunogen and has been included in nearly all vaccine formulations tested so far (28, 30, 31). Env surface (SU) and transmembrane glycoproteins (gp) are actively targeted by the immune system (9, 10, 47), and Env-specific antibodies and cytotoxic T lymphocytes (CTLs) are produced early in infection. The appearance of these effectors also coincides with the decline of viremia during the acute phase of infection (30, 32). Individuals who control HIV infection in the absence of antiretroviral therapy have Env-specific NA and CTL responses that are effective against a wide spectrum of viral strains (14, 23, 35, 52, 60). At least some of the potentially protective epitopes in Env appear to interact with the cellular receptors during viral entry and are therefore highly conserved among isolates (31, 33, 39, 63). However, these epitopes have complex secondary and tertiary structures and are only transiently exposed by the structural changes that occur during the interaction between Env and its receptors (10, 11, 28). As a consequence, these epitopes are usually concealed from the immune system, and this may explain, at least in part, why Env-based vaccines have failed to show protective efficacy. Indeed, data from previous studies suggested that protection may be most effectively triggered by nascent viral proteins (22, 28, 30, 48, 62).We have conducted a proof-of-concept study to evaluate whether presenting Env to the immune system in a manner as close as possible to what occurs in the context of a natural infection may confer some protective advantage. The study was carried out with feline immunodeficiency virus (FIV), a lentivirus similar to HIV that establishes persistent infections and causes an AIDS-like disease in domestic cats. As far as it is understood, FIV evades immune surveillance through mechanisms similar to those exploited by HIV, and attempts to develop an effective FIV vaccine have met with difficulties similar to those encountered with AIDS vaccines (25, 37, 66). In particular, attempts to use FIV Env as a protective immunogen have repeatedly failed (13, 38, 58). Here we report the result of one experiment in which specific-pathogen-free (SPF) cats primed with a DNA immunogen encoding FIV Env and feline granulocyte-macrophage colony-stimulating factor (GM-CSF) and boosted with viable, autologous T lymphocytes ex vivo that were transduced to express Env and feline interleukin 15 (IL-15) showed a remarkable level of protection against challenge with ex vivo FIV. Consistent with recent findings indicating the importance of NA in controlling lentiviral infections (1, 59, 63), among the immunological parameters investigated, only the titers of NA correlated inversely with protection. Collectively, the findings support the notion that Env is a valuable vaccine immunogen but needs to be administered in a way that permits the expression of its full protective potential.     

HIV-1 Resistance to CCR5 Antagonists Associated with Highly Efficient Use of CCR5 and Altered Tropism on Primary CD4+ T Cells

We previously reported on a panel of HIV-1 clade B envelope (Env) proteins isolated from a patient treated with the CCR5 antagonist aplaviroc (APL) that were drug resistant. These Envs used the APL-bound conformation of CCR5, were cross resistant to other small-molecule CCR5 antagonists, and were isolated from the patient''s pretreatment viral quasispecies as well as after therapy. We analyzed viral and host determinants of resistance and their effects on viral tropism on primary CD4⁺ T cells. The V3 loop contained residues essential for viral resistance to APL, while additional mutations in gp120 and gp41 modulated the magnitude of drug resistance. However, these mutations were context dependent, being unable to confer resistance when introduced into a heterologous virus. The resistant virus displayed altered binding between gp120 and CCR5 such that the virus became critically dependent on the N′ terminus of CCR5 in the presence of APL. In addition, the drug-resistant Envs studied here utilized CCR5 very efficiently: robust virus infection occurred even when very low levels of CCR5 were expressed. However, recognition of drug-bound CCR5 was less efficient, resulting in a tropism shift toward effector memory cells upon infection of primary CD4⁺ T cells in the presence of APL, with relative sparing of the central memory CD4⁺ T cell subset. If such a tropism shift proves to be a common feature of CCR5-antagonist-resistant viruses, then continued use of CCR5 antagonists even in the face of virologic failure could provide a relative degree of protection to the T_CM subset of CD4⁺ T cells and result in improved T cell homeostasis and immune function.Entry of human immunodeficiency virus (HIV) into target cells is a complex, multistep process that is initiated by interactions between the viral envelope (Env) protein gp120 and the host cell receptor CD4, which trigger conformational changes in gp120 that form and orient the coreceptor binding site (9, 24). Upon binding to coreceptor, which is either CCR5 or CXCR4 for primary HIV isolates, Env undergoes further conformational changes resulting in insertion of the gp41 fusion peptide into the host cell membrane and gp41-mediated membrane fusion (8, 15, 26). Targeting stages of the HIV entry process with antiretroviral drugs is a productive method of inhibiting HIV replication, as demonstrated by the potent antiviral effects of small-molecule CCR5 antagonists and fusion inhibitors (23, 35, 49). As with other antiretroviral drugs, HIV can develop resistance to entry inhibitors, and a detailed understanding of viral and host determinants of resistance will be critical to the optimal clinical use of these agents.The coreceptor binding site that is induced by CD4 engagement consists of noncontiguous regions in the bridging sheet and V3 loop of gp120 (4, 18, 42, 43, 50). Interactions between gp120 and CCR5 occur in at least two distinct areas: (i) the bridging sheet and the stem of the V3 loop interact with sulfated tyrosine residues in the N′ terminus of CCR5, and (ii) the crown of the V3 loop is thought to engage the extracellular loops (ECLs), particularly ECL2, of CCR5 (10-12, 14, 18, 28). Small-molecule CCR5 antagonists bind to a hydrophobic pocket in the transmembrane helices of CCR5 and exert their effects on HIV by altering the position of the ECLs, making them allosteric inhibitors of HIV infection (13, 31, 32, 46, 52). The conformational changes in CCR5 that are induced by CCR5 antagonists vary to some degree with different drugs, as evidenced by differential binding of antibodies and chemokines to various drug-bound forms of CCR5 (47, 54).CCR5 antagonists are unusual among antiretroviral agents in that they bind to a host protein rather than a viral target, and therefore the virus cannot directly mutate the drug binding site to evade pharmacologic pressure. Nevertheless, HIV can escape susceptibility to CCR5 antagonists. One mechanism by which this occurs is the use of the alternative HIV coreceptor, CXCR4. In vivo, this has most often been manifest as the outgrowth of R5/X4-tropic HIV isolates that were present in the patient''s circulating viral swarm prior to therapy (17, 27, 55). A second mechanism of HIV resistance to CCR5 antagonists is the use of drug-bound CCR5 as a coreceptor for entry. Resistant viruses that utilize drug-bound CCR5 have been identified following in vitro passaging with multiple CCR5 antagonists (1, 2, 22, 33, 36, 51, 56). Recently, we identified a panel of viral Envs able to use aplaviroc (APL)-bound CCR5 that were isolated from a patient (21, 48). The Envs from this patient were cross resistant to the CCR5 antagonists AD101, TAK779, SCH-C, and maraviroc. Surprisingly, this antiretroviral-naïve patient harbored Envs resistant to aplaviroc prior to the initiation of therapy. In the present study, we have examined viral and host factors that contribute to aplaviroc resistance and examined the consequences of resistance for viral tropism. Aplaviroc resistance determinants were located within the V3 loop of gp120, although additional residues diffusely spread throughout the gp120 and gp41 proteins modulated the magnitude of drug resistance. The resistant virus displayed altered interactions between gp120 and CCR5 such that the virus became critically dependent upon the N′ terminus of drug-bound CCR5. This differential recognition of CCR5 in the presence of aplaviroc was also associated with increased dependence on a higher CCR5 receptor density for efficient virus infection and a tropism shift toward effector memory cells on primary CD4⁺ T cells.